Properties of Cholinesterases
Cholinesterases (ChEs) are highly polymorphic carboxylesterases of broad substrate specificity, involved in the termination of neurotransmission in cholinergic synapses and neuromuscular junctions. ChEs terminate the electrophysiological response to the neurotransmitter acetycholine (ACh) by degrading it very rapidly (1). ChEs belong to the B type carboxylesterases on the basis of their sensitivity to inhibition by organophosphorous (OP) poisons (2) and are primarily classified according to their substrate specificity and sensitivity to selective inhibitors into acetylcholinesterase (ACHE, acetylcholine acetylhydrolase, EC 3.1.1.7) and butyrylcholinesterase (BuChE, acylcholine acylhydrolase, EC 3.1.1.8) (3). Further classifications of ChEs are based on their charge, hydrophobicity, interaction with membrane or extracellular structures and multisubunit association of catalytic and non-catalytic "tail" subunits (4,5).
The severe clinical symptoms resulting from OP intoxication (6) are generally attributed to their inhibitory interaction on AChE (7). OPs are substrate analogues to ChEs. The labeled OP diisopropylfluorophosphate (DFP) was shown to bind convalently to the serine residue at the active esteratic site region of ChEs, that is common to all of the carboxyl-esterases (8,9). However, the binding and inactivation capacity of OPs on ChEs is considerably higher than their effect on other serine hydrolases. Furthermore, even within species the inhibition of ChEs by different OPs tends to be highly specific to particular ChE types (10). In order to improve the designing of therapeutic and/or prophylactic drugs to OP intoxication, it was therefore desirable to reveal the primary amino acid sequence and three dimensional structure of human ACHE, and to compare them to those of human BuChE, as well as to the homologous domains in other serine hydrolases.
AChE may be distinguished from the closely related enzyme BuChE by its high substrate specificity and sensitivity to selective inhibitors (11). Both enzymes exist in parallel arrays of multiple molecular forms, composed of different numbers of catalytic and non-catalytic subunits (12). However, in humans, as in other species, they display a tissue-specific mode of expression. BuChE, assumed to be produced in the liver, is the principal species in serum (13). In contrast, AChE is the major cholinesterease in various human brain regions (14), including the cholinoceptive basal brain ganglia (15).
Extensive research efforts by several groups resulted in recent years in the isolation of cDNA clones encoding the electric fish AChE (16,17), Drosophila AChE (18,19) and human BuChE (20,21). However, the primary structure of mammalian, and more particularly, human AChE remained unknown.
Interaction of Cholinesterases with Organophosphorous Insecticides and War Gases
The use of organophosphorous (OP) anticholinesterase compounds in war (22) and as agricultural insecticides (23) resulted, over the last 40 years, in an interesting number of cases of acute and delayed intoxication. These included damage to the peripheral and central nervous system, myopathy, psychosis, general paralysis and death (24). Estimations are that 19,000 deaths occur out of the 500,000 to 1 million annual pesticide-associated poisonings (25). Previous animal studies demonstrated that methyl parathion administration suppressed growth and induced ossification in both mice and rats, as well as high mortality and cleft palate in the mouse (26). In humans, malformations of the extremities and fetal death were correlated with exposure to methyl parathion in 18 cases (27). In addition, a neonatal lethal syndrome of multiple malformations was reported in women exposed to unspecific insecticides during early pregnancy (28).
Complete inhibition of ChEs by the administration of OP poisons is lethal (6). This inhibition is achieved by formation of a stable stoichiometric (1:1) covalent conjugate with the active site serine (7), followed by a parallel competing reaction, termed "aging", which transforms the inhibited ChE into a form that cannot be generated by the commonly used reactivators (7) such as active-site directed nucleophiles (e.g., quaternary oximes) which detach the phosphoryl moiety from the hydroxyl group of the active site serine (70). The aging process is believed to involve dealkylation of the covalently bound OP group (7), and renders therapy of intoxication by certain organophosphates such as Sarin, DFP and Soman, exceedingly difficult (29).
Use of preparations comprising ChEs for therapeutical purposes has been demonstrated to be effective at laboratory level: purified AChE from fetal calf serum has been shown to protect rats from 2 lethal doses of Soman (a war OP poison) with half life of 5-6 days (37,38). Purified BuChE from human serum has been shown to improve the symptoms of OP-intoxicated patients (31).
Interaction of Cholinesterases with Succinylcholine--Post-Operative Apnea
Succinylcholine which acts as a competitive analogue of acetylcholine, is often used in surgery as a short-term muscle relaxant. Since the drug is hydrolyzed by BuChE, its administration into individuals carrying genetically abnormal BuChE causes prolonged apnea (32). The most common variant with this problem is the atypical variant E.sup.s, for which 3-6% of the Caucasian population is heterozygous and about 0.05% is homozygous (33). This enzyme hydrolyzes acetylcholine but not succinylcholine (34). Another variant, E.sup.1, which causes the complete absence of catalytically active serum BuChE in homozygotes, is also associated with this clinical problem (35). This type of "silent" enzyme cannot hydrolyze any ChE substrate, nor can it bind organophosphate compounds (9). High frequency of atypical and silent BuChE genes was reported among Iraqui and Iranian Jews (11.3% for heterozygotes and 0.08% for homozygotes, respectively) (36-38). This could explain the high frequency of reports of prolonged apnea following surgery in Israel, and apparently in many other countries. It is likely that AChE could be administered to patients to rid the body of the succinylcholine in cases of prolonged apnea.
Alterations in the Level and Properties of Cholinesterases
In several neurological or genetic disorders, such as Senile Dementia of the Alzheimer's type or Down's syndrome, modification in both the level (39) and the composition of molecular forms (40) of human brain acetylcholinesterase have been reported. In the Alzheimer's disease, the levels of AChE in cholinergic brain areas drops by about 50% and the tetrameric form of the enzyme dissappears completely. Individuals with Down's syndrome invariably develop manifestations of the Alzheimer's disease before the age of 40. In addition, it has been observed that neural tube defects in human embryos are clinically characterized by secretion of AChE tetramers into the amniotic fluid. These phenomenae are currently tested for by sucrose gradient fractionation, followed by enzymatic assays of substrate hydrolysis or gel electrophoresis and AChE activity staining. Simple and selective quantitative assays for specific AChE forms have not yet been developed.
Furthermore, death at very early stages of development has been observed in Homozygote Drosophila mutants lacking the Ace locus which controls AChE biosynthesis and in nematode mutants defective in the expression of their four ChE genes. It is very likely that homozygous mutations in AChE genes in humans will result in early abortion or in severe neurological and possibly other malformations in the fetus. No methods to determine whether specific individuals carry such mutations have been disclosed so far.
Relationship between Cholinesterases and Hematopoiesis and Blood Cells Differentiation
Biochemical and histochemical analyses indicate that both acetylcholinesterase and butyrylcholinesterase are expressed in high levels in various fetal tissues of multiple eukaryotic organisms (41), where ChE are coordinately regulated with respect to cell proliferation and differentiation (42). However, no specific role could be attributed to ChE in embryonic development and their biological function(s) in these tissues remained essentially unknown (71).
In addition to its presence in the membrane of mature erythrocytes, AChE is also intensively produced in developing blood cells in vivo (43) and in vitro (44) and its activity serves as an accepted marker for developing mouse megakaryocytes (45). Furthermore, administration of acetylcholine analogues as well as ChE inhibitors has been shown to induce megakaryocytopoiesis and increased platelet counts in the mouse (46), implicating this enzyme in the commitment and development of these hematopoietic cells.
Recently, the cDNA coding for BuChE has been cloned (20) and BuChEcDNA hybridizing sequences have been localized to chromosome sites 3q21,26 and 16q12 (47). It is of importance to emphasize that the chromosome 3q21,26 region includes breakpoints that were repeatedly observed in peripheral blood chromosomes of patients with acute myelodisplastic leukemia (AML) (48,49). These cases all featured enhanced megakaryocytopoiesis, high platelet count and rapid progress of the disease (15). Accumulating evidence in recent reports implicates chromosomal breakpoints with molecular changes in the structure of DNA and the induction of malignancies (51). Therefore, the connection between: (a) abnormal control of megakaryocytopoiesis in AML as well as in mouse bone-marrow cells subjected to ChE inhibition; (b) cholinesterase genes location on the long arm of chromosome 3; and (c) chromosomal aberrations in that same region in AML, appeared more than coincidental (for discussion see (47)).
The putative correlation between the human genes coding for ChEs and the regulation of megakaryocytopoiesis has been examined by searching for structural changes in the human AChE and ChE genes from peripheral blood DNA in patients with leukemia, platelet count abnormalities, or both. Proof of the active role of these enzymes in the progress of human hematopoiesis had to be established.
Relationship between Cholinesterases and Ovarian Carcinomas
High level of expression of AChE and ChE in tumors was reported in the past (66,67), however, it was still to be elucidated whether this high expression level is effected by gene amplification. The rapidly progressing carcinomas of the ovary (68) may offer a promising model in which to test said possibility since sections from these tumors exhibit pronounced diffuse cytochemical staining of ChE activities (66), whereas ChE expression in normal ovarian tissue appears to be confined to maturing oocytes (47).
The possible amplification of the human AChE and ChE genes in primary ovarian carcinomas, and their expression in dividing cells within tumor loci, implicating involvement of cholinesterase in tumor growth and development, had to be established.